The use of impedance analysis for monitoring microbiological effects is well known.
K. Sachsenheimer, L. Pires, M. Adamek, Th. Schwartz and B. E. Rapp: Monitoring Biofilm Growth using a Scalable Multichannel Impedimetric Biosensor, in: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Oct. 2-6, 2011, Seattle, Wash., USA, pages 1968 to 1970 discloses a multichannel electrochemical impedance spectroscopy (EIS) based biosensor that allows the monitoring of biofilm growth. The bacterial strains (Pseudomonas aeruginosa, Stenotrophomonas maltophilia) were monitored for up to 96 hours with the biofilm directly growing on an electrode structure. Said electrode structure comprises a working electrode and a counter electrode on a substrate. The biofilm growth on the electrode structure hinders the charge transfer between the electrodes and therefore increases the measured impedance. The impedance increases over time because of a biofilm growing on the electrode surface. Two electrodes are used to compensate drift effects, with the measurement electrode probe carrying the bacteria and a reference electrode being only exposed to feeding medium.
WO 2005/047482 A2 and WO 2005/077104 A2 disclose a real time electronic cell sensing system comprising an electrode structure and a plurality of receptacles placed on top of the electrode structure. Two or more electrode arrays are fabricated on a non-conducting substrate. The substrate has a surface suitable for cell attachment or growth. The cell attachment or growth on said substrate results in a detectable change in impedance between the electrode structures within each electrode array. For measurement of the cell-substrate impedance, an impedance analyzer is connected to the connection pads of the substrate for measuring the impedance values at specific frequencies.
The impedance is a complex value comprising the ohmic resistance and the reactance. The reactance is the imaginary part of the impedance and provides the value of the capacitance of the electrode structure. Cell Index values are calculated from the measured impedance data. The dimensionless cell index measures the relative change in the electrical impedance at certain frequency (fn). The Cell Index at a given time point t (CI(t)) is calculated as follows:
      CI    ⁢                  ⁢          (      t      )        =                    R        ⁢                                  ⁢                  (                                    f              n                        ,            t                    )                    -              R        ⁢                                  ⁢                  (                                    f              n                        ,                          t              0                                )                            Z      n      
where
fn is the frequency that impedance measurement is carried out,
R(fn, t) is the measured impedance at frequency fn at time point t,
R(fn, t0) is the measured impedance at frequency fn at time point t0, usually t0 is the time when the background is measured),
Zn is the corresponding frequency factor of fn.
For example, the xCELLigence® system, which is available from the company ACEA Biosciences, Inc. USA, measures impedance at three discrete frequencies, i.e., f1=10 kHz, f2=25 kHz, and f3=50 kHz. The corresponding frequency factors are Z1=15 Ohm, Z2=12 Ohm, and Z3=10 Ohm, respectively.
The resistance and reactance part of the cell index can be obtained by mathematical transformations. In another example, the cell index can be calculated at each measured frequency by dividing the resistance value and/or the reactance value of the electrode arrays when cells are present on, or attached to the electrodes by the baseline resistance and/or reactance. Hereby, the maximum value in the reactance ratio over the frequencies spectrum can be found or determined. Alternatively a specific value, e.g. the value 1 or the measured value at the start of the experiment, can be subtracted from the value in the reactance ratio.
Further examples for determining the cell index are disclosed in the references WO 2005/047482 A2 and WO 2005/077104 A2 cited above.
WO 2004/010102 A2 discloses an impedance-based apparatus for analyzing cells and particles comprising an upper chamber adapted to receive and retain a cell sample, a lower chamber having at least two electrodes, and a biocompatible porous membrane having a porosity sufficient to allow cells to migrate therethrough. The cells migrating to the lower chamber attach to the electrodes. In another example the microporous membrane and the electrode structure is placed between the upper and lower chamber. Again cells are attaching to the electrode structure and growing on the electrode structure. With increasing cell numbers, the impedance at specific frequencies and the calculated cell index is increasing. Thus, proportionally the capacity decreases with increasing cells adhering on the electrode structure.
Said disclosed cell monitoring apparatus is commercial available under the trademark xCELLigence® RTCA with CIM-Plates and E-plates. Said device is available from the company ACEA Biosciences, Inc. USA.